Anti-HER2 Cancer-Specific mAb, H2Mab-250-hG1, Possesses Higher Complement-Dependent Cytotoxicity than Trastuzumab

Cancer-specific monoclonal antibodies (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy are innovative therapeutic strategies for minimizing adverse effects. We previously established a cancer-specific anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody (mAb), H2Mab-250/H2CasMab-2. In flow cytometry and immunohistochemistry, H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, strongly recognizes both breast cancer and normal epithelial cells in flow cytometry. The human IgG1 version of H2Mab-250 (H2Mab-250-hG1) possesses compatible in vivo antitumor effects against breast cancer xenografts to trastuzumab despite the lower affinity and effector activation than trastuzumab in vitro. This study compared the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC) between H2Mab-250-hG1 and trastuzumab. Both H2Mab-250-hG1 and trastuzumab showed ADCC activity against HER2-overexpressed Chinese hamster ovary -K1 and breast cancer cell lines (BT-474 and SK-BR-3) in the presence of human natural killer cells. Some tendency was observed where trastuzumab showed a more significant ADCC effect compared to H2Mab-250-hG1. Importantly, H2Mab-250-hG1 exhibited superior CDC activity in these cells compared to trastuzumab. Similar results were obtained in the mouse IgG2a types of both H2Mab-250 and trastuzumab. These results suggest the different contributions of ADCC and CDC activities to the antitumor effects of H2Mab-250-hG1 and trastuzumab, and indicate a future direction for the clinical development of H2Mab-250-hG1 against HER2-positive tumors.


Introduction
Human epidermal growth factor receptor 2 (HER2) is a member of the receptor tyrosine kinases.Heterodimerization of HER2 with other HER family members and the ligands or ligand-independent HER2 homodimerization results in the autophosphorylation of the cytoplasmic domain.This event initiates a variety of signaling, such as the RAS and PI3K pathways, leading to cancer cell proliferation, survival, and invasiveness [1].The overexpression of HER2 is observed in approximately 20% of breast cancers [2] and 20% of gastric cancers [3], which are associated with higher rates of recurrence and shorter overall survival.
Trastuzumab, an anti-HER2 monoclonal antibody (mAb), exhibited in vitro antiproliferative efficacy and a potent antitumor effect in vivo [4,5].The combination of chemotherapy with trastuzumab in HER2-positive breast cancer patients with metastasis improves the progression-free survival and overall survival [6].Trastuzumab was approved by the U.S. Food and Drug Administration (FDA) for the treatment of HER2positive breast cancer [6] and has been the most effective therapy for it for more than 20 years [7].Trastuzumab is administered in patients with HER2-overexpressed tumors, which are defined by solid and complete membranous staining of more than 10% of cells in immunohistochemistry (IHC 3+) and/or in situ hybridization (ISH)-amplified [8].Furthermore, trastuzumab-deruxtecan (T-DXd), a trastuzumab-based antibody-drug conjugate (ADC), has been developed and approved by the FDA [9].T-DXd exhibited superior efficacy in not only HER2-positive breast cancers [10,11] but also HER2-low (IHC 1+ or IHC 2+/ISH-non-amplified) advanced breast cancers [12] and HER2-mutant lung cancers [13].
Because half of all breast cancers are classifiable as HER2-low, a significant number of patients are estimated to receive the benefit from T-DXd therapy [14].
The immunologic engagement of trastuzumab mediates the clinical efficacy [4].Antibodydependent cellular cytotoxicity (ADCC) is elicited by natural killer (NK) cells or macrophages upon the binding of Fcγ receptors (FcγRs) to the Fc region of mAbs [4].Trastuzumab is a humanized IgG 1 mAb that binds to FcγRs [15] and activates macrophages, neutrophils, and dendritic cells, which change the adaptive immunity by cytokine production, chemotaxis, and antigen presentation [4].Moreover, the FcγR binding results in the activation of NK cells and macrophages, which can result in the target cell killing [4].However, the ADCC is impaired by the N-linked glycosylation in the Fc region [16].In particular, a lack of core fucose on the Fc N-glycan enhances the Fc binding to the FcγRs on effector cells [17].Therefore, a core fucose deficiency on the Fc N-glycan has been shown to enhance the binding to FcγR on effector cells [17] and exert potent antitumor effects [18].The defucosylated recombinant mAbs can be produced using fucosyltransferase 8-knockout Chinese hamster ovary (CHO) cells [19].
Complement-dependent cellular cytotoxicity (CDC) is also exerted by the Fc domain of mAbs [20,21].Although complements have been thought of as an adjunctive component of the antibody-mediated cytolytic effects, complement is currently considered an essential effector of the tumor cytotoxic responses of mAb-based immunotherapy [21].Through the development of a chimeric anti-CD20 mAb, rituximab, for the treatment of B cell lymphomas, the involvement with the cytolytic capacity of the complement was revealed in the antitumor effect [22,23].In not only anti-CD20 but also anti-CD38 and CD52 immunotherapies, the cytolytic capacity of the tumor by complements has been shown [23][24][25].Furthermore, a growing body of evidence suggests that complements play crucial functions in not only tumor cytolysis but also several immunologic roles in antitumor immunity [26,27].The crosstalk of complement effectors and cellular signaling pathways influence the T and B cell responses, T helper/effector T cell survival, differentiation, and B cell activation.
A common adverse effect of anti-HER2 mAbs and the ADCs is cardiotoxicity [28].Routine cardiac monitoring is required for patients [29].Moreover, the lack of cardiac trabeculae is observed in ErbB2 (ortholog of HER2)-knockout mice [30], and the features of dilated cardiomyopathy are observed in ventricular-specific ErbB2-knockout mice [31].These results indicate that HER2 is involved in normal heart development and homeostasis.Therefore, more selective or specific anti-HER2 mAbs against tumors are required to reduce heart failures.
This study compared the ADCC and CDC between H 2 Mab-250 and trastuzumab against HER2-overexpressed CHO-K1 (CHO/HER2) and breast cancer cell lines.

ADCC and CDC by H 2 Mab-250 and Trastuzumab against CHO/HER2
To confirm the requirement of HER2 in the ADCC and CDC of H 2 Mab-250 and trastuzumab, we used CHO-K1 and CHO/HER2 and performed the ADCC and CDC assays.CHO/HER2 was also recognized by H 2 Mab-250 and trastuzumab with low and high reactivity, respectively [34].As shown in Figure 2A, both H 2 Mab-250-hG 1 and trastuzumab induced ADCC against CHO/HER2 cells (70% and 77% cytotoxicity, respectively) more effectively than the control human IgG (13% cytotoxicity; p < 0.01).In contrast, H 2 Mab-250-hG 1 showed a significant CDC (63% cytotoxicity) compared to the control human IgG (10% cytotoxicity; p < 0.01, Figure 2B).However, trastuzumab did not show a significant CDC (Figure 2B).In CHO-K1, we did not observe ADCC and CDC in the presence of H 2 Mab-250 and trastuzumab (Figure 2C,D).These results indicate that the recognition of HER2 is essential to the exertion of ADCC and CDC by H 2 Mab-250-hG 1 and trastuzumab.Furthermore, dose-dependent activation of CDC activity by H 2 Mab-250 and trastuzumab was observed against CHO/HER2, but not CHO-K1 (Figure 2E).H 2 Mab-250-hG 1 exhibited a significant CDC compared to the control human IgG from 25 µg/mL and showed it compared to trastuzumab at 100 µg/mL (Figure 2E).

ADCC and CDC by H2Mab-250 and Trastuzumab against CHO/HER2
To confirm the requirement of HER2 in the ADCC and CDC of H2Mab-250 and trastuzumab, we used CHO-K1 and CHO/HER2 and performed the ADCC and CDC assays.CHO/HER2 was also recognized by H2Mab-250 and trastuzumab with low and high reactivity, respectively [34].As shown in Figure 2A, both H2Mab-250-hG1 and trastuzumab induced ADCC against CHO/HER2 cells (70% and 77% cytotoxicity, respectively) more effectively than the control human IgG (13% cytotoxicity; p < 0.01).In contrast, H2Mab-250-hG1 showed a significant CDC (63% cytotoxicity) compared to the control human IgG (10% cytotoxicity; p < 0.01, Figure 2B).However, trastuzumab did not show a significant CDC (Figure 2B).In CHO-K1, we did not observe ADCC and CDC in the presence of H2Mab-250 and trastuzumab (Figure 2C,D).These results indicate that the recognition of HER2 is essential to the exertion of ADCC and CDC by H2Mab-250-hG1 and trastuzumab.Furthermore, dose-dependent activation of CDC activity by H2Mab-250 and trastuzumab was observed against CHO/HER2, but not CHO-K1 (Figure 2E).H2Mab-250-hG1 exhibited a significant CDC compared to the control human IgG from 25 µg/mL and showed it compared to trastuzumab at 100 µg/mL (Figure 2E).

Antitumor Activities by H 2 Mab-250-hG 1 and Trastuzumab
Next, we examined the in vivo antitumor efficacy of H 2 Mab-250-hG 1 and trastuzumab in the CHO/HER2 xenograft model.We injected H 2 Mab-250-hG 1 , trastuzumab, and control human IgG intraperitoneally on days 7, 14, and 21 after inoculating CHO/HER2.Furthermore, human NK cells were injected around the tumors on the same days of the Abs injection.Following the inoculation, we measured the tumor volume on days 7, 14, 21, and 28.The H 2 Mab-250-hG 1 and trastuzumab administration led to a significant and similar reduction in the CHO/HER2 xenograft on day 28 (p < 0.01) compared with that of the control (Figure 3A).Both H 2 Mab-250-hG 1 and trastuzumab administration resulted in an 81% reduction in the CHO/HER2 xenograft volume compared with the control human IgG on day 28.treated CHO/HER2 xenograft-bearing mice (Figure 3D), and there was no difference in the body appearance in those mice (Figure 3E).
We also investigated the pharmacokinetics of H2Mab-250-hG1 and trastuzumab after administration in nude mice.As shown in Supplementary Figure S2, the half-lives of H2Mab-250-hG1 and trastuzumab were determined as 128 and 133 h, respectively.These results indicate that H2Mab-250-hG1 possesses a similar half-life compared to trastuzumab.The CHO/HER2 xenografts from the H 2 Mab-250-hG 1 -and trastuzumab-treated mice weighed significantly less than those from the control human IgG-treated mice (93% and 94% reduction, respectively; p < 0.05, Figure 3B,C).There was no significant difference between the H 2 Mab-250-hG 1 -and trastuzumab-treated xenografts.
A body weight loss was not observed in the H 2 Mab-250-hG 1 -and trastuzumab-treated CHO/HER2 xenograft-bearing mice (Figure 3D), and there was no difference in the body appearance in those mice (Figure 3E).
We also investigated the pharmacokinetics of H 2 Mab-250-hG 1 and trastuzumab after administration in nude mice.As shown in Supplementary Figure S2, the half-lives of H 2 Mab-250-hG 1 and trastuzumab were determined as 128 and 133 h, respectively.These results indicate that H 2 Mab-250-hG 1 possesses a similar half-life compared to trastuzumab.

Antitumor Activities by Mouse IgG2a-Type H2Mab-250 and Trastuzumab
Next, we examined the in vivo antitumor efficacy of H2Mab-250-mG2a and tras-mG2a in the CHO/HER2 xenograft model.We injected H2Mab-250-mG2a, tras-mG2a, and a control mouse IgG2a intraperitoneally on days 9 and 16 after inoculating CHO/HER2.Following the inoculation, we measured the tumor volume on days 9, 16, and 21.The H2Mab-250-mG2a and tras-mG2a administration led to a potent and similar reduction in the CHO/HER2 xenograft on days 16 and 21 (p < 0.01) compared with that of the control mouse IgG2a (Figure 5A).The H2Mab-250-mG2a and tras-mG2a administration resulted in a 77% and 74% reduction in the CHO/HER2 xenograft volume compared with the control mouse IgG2a on day 21.These results indicate that tras-mG 2a showed a superior ADCC compared to H 2 Mab-250-mG 2a .In contrast, H 2 Mab-250-mG 2a exhibited a superior CDC to CHO/HER2 compared to tras-mG 2a.

Antitumor Activities by Mouse IgG 2a -Type H 2 Mab-250 and Trastuzumab
Next, we examined the in vivo antitumor efficacy of H 2 Mab-250-mG 2a and tras-mG 2a in the CHO/HER2 xenograft model.We injected H 2 Mab-250-mG 2a , tras-mG 2a , and a control mouse IgG 2a intraperitoneally on days 9 and 16 after inoculating CHO/HER2.Following the inoculation, we measured the tumor volume on days 9, 16, and 21.The H 2 Mab-250-mG 2a and tras-mG 2a administration led to a potent and similar reduction in the CHO/HER2 xenograft on days 16 and 21 (p < 0.01) compared with that of the control mouse IgG 2a (Figure 5A).The H 2 Mab-250-mG 2a and tras-mG 2a administration resulted in a 77% and 74% reduction in the CHO/HER2 xenograft volume compared with the control mouse IgG 2a on day 21. was observed between the control and H2Mab-250-mG2a-treated mice.Those mice had no difference in body appearance (Figure 5E).

Comparison of Antitumor Activities by H2Mab-250-hG1 and Trastuzumab in the Absence of Human NK Cells
As shown in Figure 3, we injected human NK cells with H2Mab-250-hG1 and trastuzumab because high ADCC activity was expected.Since H2Mab-250-hG1 possesses a higher CDC activity, we next compared the antitumor effects of H2Mab-250-hG1 and trastuzumab without the human NK cells.We injected H2Mab-250-hG1 and trastuzumab intraperitoneally on days 7, 14, and 21 after inoculating CHO/HER2.Following the inoculation, we measured the tumor volume on days 7, 10, 14, 16, 21, 24, and 29.We observed the more potent antitumor efficacy of H2Mab-250-hG1 in relation to both the tumor volume (p < 0.01, Figure 6A) and weight (p < 0.01, Figure 6B) compared with that of trastuzumab at day 29 (Figure 6C).
Figure 5D shows that body weight loss was not observed in the H 2 Mab-250-mG 2aand tras-mG 2a -treated CHO/HER2 xenograft-bearing mice.However, a slight difference was observed between the control and H 2 Mab-250-mG 2a -treated mice.Those mice had no difference in body appearance (Figure 5E).

Comparison of Antitumor Activities by H 2 Mab-250-hG 1 and Trastuzumab in the Absence of Human NK Cells
As shown in Figure 3, we injected human NK cells with H 2 Mab-250-hG 1 and trastuzumab because high ADCC activity was expected.Since H 2 Mab-250-hG 1 possesses a higher CDC activity, we next compared the antitumor effects of H 2 Mab-250-hG 1 and trastuzumab without the human NK cells.We injected H 2 Mab-250-hG 1 and trastuzumab intraperitoneally on days 7, 14, and 21 after inoculating CHO/HER2.Following the inoculation, we measured the tumor volume on days 7, 10, 14, 16, 21, 24, and 29.We observed the more potent antitumor efficacy of H 2 Mab-250-hG 1 in relation to both the tumor volume (p < 0.01, Figure 6A) and weight (p < 0.01, Figure 6B) compared with that of trastuzumab at day 29 (Figure 6C).

Discussion
We have developed CasMabs against HER2 (H 2 Mab-250 [33,34]), podocalyxin (PcMab-6 [35]), and podoplanin (LpMab-2 [36] and LpMab-23 [37]) by evaluating the reactivity against cancer and normal cells in flow cytometry and immunohistochemistry.We also showed the in vivo antitumor effect of the recombinant mAbs (mouse IgG 2a or human IgG 1 types) derived from the abovementioned mAbs [32][33][34].Especially, H 2 Mab-250 showed a potent antitumor effect in vivo [34] despite the lower reactivity and affinity than trastuzumab in vitro [33].However, the reason for this has not been clarified.In this study, we compared the ADCC and CDC activity of H 2 Mab-250 and trastuzumab and found that H 2 Mab-250 exhibited a superior CDC activity against breast cancer and HER2-overexpressed cells compared to trastuzumab (Figures 1, 2 and 4).Furthermore, both H 2 Mab-250-hG 1 and H 2 Mab-250-mG 2a showed compatible antitumor effects compared to the corresponding isotype of trastuzumab (Figures 3 and 5).These results suggest that the CDC activity of H 2 Mab-250 would compensate for the lower ADCC activity in the antitumor efficacy.
Complement is an essential effector of tumor cytotoxic responses in mAb-based immunotherapy [21].The engagement of the Fc domain of mAbs with complement C1q triggers the assembly of the active C1 complex (C1q, C1r, and C1s), which initiates the cascade.The downstream activation of terminal complement components results in the assembly of the pore-forming membrane attack complex (MAC or C5b-C9) on the tumor cell membrane, which promotes the terminal lytic pathway [21].Complement activation also leads to tumor cell opsonization by C3-derived opsonins (C3b, iC3b, and C3dg), which bind to the CR3/CR4 complement receptors on phagocytes (neutrophils and macrophages) and augment the FcγR-dependent phagocytic uptake of opsonized tumor cells.Furthermore, complement activation generates pro-inflammatory mediators (C3a and C5a).The anaphylatoxin C5a upregulates the FcγRs on phagocytes and primes them for enhanced phagocytosis and increasing the magnitude of the tumor cytolytic response [21].
Because H 2 Mab-250 showed increased CDC activity only in the presence of complement (Figures 1, 2 and 4), the assembly of MAC is thought to be efficiently formed on the cells.Furthermore, the predisposition to CDC of H 2 Mab-250 is independent of the isotype or species of mAbs (Figures 2 and 4).Therefore, the complementarity-determining region and epitope of H 2 Mab-250 are thought to be necessary.In Figure 6, the antitumor effects of H 2 Mab-250-hG 1 were higher than those of trastuzumab without human NK cells, indicating that H 2 Mab-250-hG 1 exerts antitumor activities with much higher CDC than trastuzumab in vivo.Several factors, including the antigen size and density, determine the engagement of the classical complement pathway.In addition, a geometry of the antigen-mAb complex allows efficient C1q binding [38].Furthermore, IgG antibodies can form ordered hexamers upon binding to their antigen on cell surfaces.These hexamers efficiently bind the hexavalent complement component C1q, the first step in the classical pathway of complement activation [39,40].The structure of the H 2 Mab-250-HER2 complex may provide adequate access for complements to exert CDC.Further investigation and confirmation are required to clarify the mechanisms of CDC in H 2 Mab-250.
Trastuzumab exerts antitumor activity through multiple mechanisms of action but is incapable of eliciting CDC in HER2-positive cancers in the presence of human serum [41,42].As shown in Figure 1B, trastuzumab did not elicit CDC against BT-474 cells.An anti-HER2 bispecific and biparatopic antibody, zanidatamab, elicited potent CDC against HER2-high tumor cells, including BT-474 cells.Zanidatamab possesses an anti-HER2-ECD4 singlechain variable fragment (scFv) linked to heavy chain 1 and an anti-HER2-ECD2 fragment antigen-binding (Fab) domain on heavy chain 2. Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization and large HER2 clusters, which are not observed with trastuzumab [43].Optimal CDC activity requires hexameric clustering of mAb Fc domains in the mAb-antigen clusters [44].We identified the epitope of H 2 Mab-250 as 613-IWKFP -617 in the HER2-ECD4.The epitope of trastuzumab is a broader sequence (residues 579-625), which includes the H 2 Mab-250 epitope [33].It is worthwhile to investigate the ability of H 2 Mab-250 to form a cluster with HER2.
Chimeric antigen receptor (CAR)-T cell therapy is rapidly advancing as a cancer treatment; however, designing an optimal CAR remains challenging.Due to the specific reactivity against cancer cells, H 2 Mab-250 is clinically developed as CAR-T cell therapy, which is evaluated in a phase I study for HER2-positive advanced solid tumors in the US (NCT06241456).We discussed the benefit of reducing CAR affinity to limit trogocytosis, which is observed in the high affinity of CAR-T cells [34].In the monotherapy of H 2 Mab-250, we have reported compatible antitumor effects against breast cancer xenograft compared to trastuzumab [32,34] and showed the importance of CDC in this study.Extensive research indicates that resistance to CDC is induced by the expression of complement regulators in tumor cells during the escape from host immune responses.Notably, an upregulation of the regulators, including CD46, CD55, and CD59, has been shown to prevent CDC through suppression of terminal complement activation and MAC assembly [45][46][47].In this regard, several strategies have been developed to overcome the resistance to CDC in mAb-based immunotherapy [48][49][50][51].Therefore, dual targeting of HER2 by H 2 Mab-250 and complement regulators should be investigated in future studies in in vitro models.

Production of Recombinant mAbs
To generate H 2 Mab-250-hG 1 , V H of H 2 Mab-250 and C H of human IgG 1 were cloned into the pCAG-Ble vector.The V L of the H 2 Mab-250 and C L of the human kappa light chain were cloned into the pCAG-Neo vector.
To generate H 2 Mab-250-mG 2a , we cloned the V H cDNA of H 2 Mab-250 and C H of mouse IgG 2a into the pCAG-Ble vector.To generate a mouse IgG 2a type of trastuzumab (tras-mG 2a ), the V H cDNA of trastuzumab and the C H cDNA of mouse IgG 2a were cloned into the pCAG-Neo vector, and the V L cDNA of trastuzumab and the C L cDNA of mouse kappa light chain were cloned into the pCAG-Ble vector.

ADCC
The ADCC of H 2 Mab-250-hG 1 and trastuzumab was measured as follows.Human NK cells were purchased from Takara Bio, Inc. (Shiga, Japan) and were used as effector cells.The NK cells were used in the following experiment immediately after thawing.We labeled the target cells (BT-474, SK-BR-3, CHO-K1, and CHO/HER2) using 10 µg/mL Calcein AM (Thermo Fisher Scientific, Inc.).The target cells were plated in 96-well plates (1 × 10 4 cells/well) and mixed with the human NK cells (effector to target ratio, 50:1) and 100 µg/mL of H 2 Mab-250-hG 1 , trastuzumab or control human IgG.The calcein release was measured after a 4.5 h incubation, and the cytotoxicity (% lysis) was calculated as described previously [34].
The ADCC of H 2 Mab-250-mG 2a and tras-mG 2a was measured as follows.Effector cells were obtained from the spleen of female BALB/c nude mice (Jackson Laboratory Japan, Inc., Kanagawa, Japan).The Calcein AM-labeled target cells (CHO-K1 and CHO/HER2) using 10 µg/mL Calcein AM were plated in 96-well plates (1 × 10 4 cells/well) and mixed with the effector cells (effector to target ratio, 50:1) with 100 µg/mL of H 2 Mab-250-mG 2a and tras-mG 2a or control mouse IgG 2a .After a 4 h incubation at 37 • C, the calcein release into the medium was measured, and the cytotoxicity (% lysis) was calculated.

CDC
The calcein-labeled target cells (BT-474, SK-BR-3, CHO-K1, and CHO/HER2) were plated and mixed with rabbit complement (final dilution 1:10, or 1:15 [Figure 2E], Low-Tox-M Rabbit Complement; Cedarlane Laboratories, Hornby, ON, Canada) and the indicated concentration of H 2 Mab-250-hG 1 , trastuzumab or control human IgG.Following incubation for 4.5 h at 37 • C, the calcein released into the medium was measured, as described above.In the case of H 2 Mab-250-mG 2a and tras-mG 2a or control mouse IgG 2a , we performed a 4 h incubation at 37 • C and the cytotoxicity (% lysis) was calculated.To examine the antitumor effect of H 2 Mab-250-hG 1 , trastuzumab, H 2 Mab-250-mG 2a , and tras-mG 2a , animal experiments were approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (approval no.2023-066 and 2023-074).We determined the body weight loss exceeding 25% and maximum tumor size exceeding 3000 mm 3 as humane endpoints.
The tumor volume was calculated using the following formula: volume = W 2 × L/2, where W is the short diameter and L is the long diameter.All the mice were euthanized by cervical dislocation.

Institutional Review Board Statement:
The Institutional Committee for Experiments of the Institute of Microbial Chemistry approved the animal experiment to examine the antitumor effect (approval no.2023-066 and 2023-074).
Informed Consent Statement: Not applicable.

Figure 1 .
Figure 1.The ADCC and CDC activities were mediated by H 2 Mab-250-hG 1 and trastuzumab in BT-474 and SK-BR-3 cells.(A,C) The ADCC induced by human NK cells in the presence of H 2 Mab-250-hG 1 , trastuzumab, or control human IgG (hIgG) against BT-474 (A) and SK-BR-3 (C) cells.(B,D) The CDC induced by complements in the presence of 100 µg/mL of H 2 Mab-250-hG 1 , trastuzumab, or control human IgG against BT-474 (B) and SK-BR-3 (D) cells.Values are shown as the mean ± SEM.Asterisks indicate statistical significance (** p < 0.01 and * p < 0.05; one-way ANOVA and Tukey's multiple comparisons test).

4. 5 .
Antitumor Activities of H 2 Mab-250-hG 1 , Trastuzumab, H 2 Mab-250-mG 2a , and Tras-mG 2a , in Tumor Xenograft Models Author Contributions: H.S., T.O. and T.T. performed the experiments.M.K.K. and Y.K. designed the experiments.H.S., T.T. and Y.K. analyzed the data.H.S. and Y.K. wrote the manuscript.All authors have read and agreed to the published version of the manuscript.